Indole-3-carbinol (I3C) derivatives and methods

ABSTRACT

The invention provides methods and compositions relating to novel bioactive derivatives of indole-3-carbinol (I3C), including pharmaceuticals comprising a pharmaceutically acceptable excipient and a compound of the general formula:  
                 
 
     which inhibits tumor cell growth. Methods of inhibiting targeted cell growth include contacting a target cell with a disclosed compound under conditions whereby the growth the target cell is inhibited, and methods for evaluating the growth inhibitory activity of the compounds include contacting a cell with an effective amount of the compound and measuring the CDK6 expression in the cell, wherein a reduction in CDK6 expression correlates with the growth inhibitory activity of the compound.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35USC120 to U.S. Ser. No.08/865,920, filed May 30, 1997, now U.S. Pat. No. 6,001,868, whichclaims priority to U.S. Ser. No. 09/425,750, filed Oct. 22, 1999, nowU.S. Pat. No. 6,150,395, which claims priority to U.S. Ser. No.09/672,641, filed Sep. 28, 2000, now U.S. Pat. No. 6,369,095.

[0002] The research carried out in the subject application was supportedin part by grants from the US Army Medical Research Command (ContractNo.RP950844). The government may have rights in any patent issuing onthis application.

FIELD OF THE INVENTION

[0003] The invention relates to indole-3-carbinols and theirbioactivity.

BACKGROUND OF THE INVENTION

[0004] More than 40,000 women die each year of metastatic breast cancerin the United States. Endocrine therapy, in particular anti-estrogentherapy, remains a major option for treatment of such patients, andresults in complete plus partial response rates of 30%. This responserate results from the fact that the growth of approximately one-third ofbreast cancers is stimulated by estrogen, which is a natural hormoneproduced in women. Treating patients with anti-estrogens, such astamoxifen, will slow the growth of these estrogen dependent tumors.Postmenopausal status, a prolonged disease-free interval, and positiveestrogen and progesterone receptors are all associated with an increasedresponse to endocrine therapy. The use of additive hormonal therapy,specifically antiestrogens, progestins, and aromatase inhibitors, havereplaced surgical ablative procedures in the majority of estrogenreceptor positive patients; with the most favorable therapeutic indexassociated with therapies that use antiestrogens (55). For patients thatare estrogen receptor negative and a subset of patients that areestrogen receptor positive, the best current approach to treating breastcancers that do not require estrogen for their growth is by surgicalremoval of the tumors. Unfortunately, even for the patients that respondto and can benefit from tamoxifen therapy, there are many detrimentalside effects including the ability of breast cancer cells to becomeresistant to tamoxifen, an increase in the potential for ovarian tumorsand an increased risk of osteoporosis. Some of these side effects aredue to tamoxifen acting as an estrogen agonist in some tissues andremaining an estrogen antagonist in other tissues.

[0005] Indole-3-carbinol (I3C) is a naturally occurring component ofBrassica vegetables, such as cabbage, broccoli, and Brussels sprouts.Exposure to dietary I3C markedly reduces the incidence of spontaneousand carcinogen-induced mammary tumors in rodents and, as disclosedherein, exhibits potent growth inhibitory activity in human breastcancer cells in vitro by inducing a G1 arrest of the cell cycle. Arecent screen of 90 potential chemopreventative agents in a series of 6short term bioassays relevant to carcinogen-induced DNA damage, tumorinitiation and promotion, and oxidative stress, revealed I3C to be oneof only 8 compounds that tested positive in all assays (1). Indeed, I3Cadministered in the diet or by oral intubation prior to treatment withcarcinogen reduced the incidence of 7,12-dimethyl-benz(α)anthracene(DMBA)-induced mammary tumors in rodents by 70-80% (2). In anotherstudy, I3C administered to rats prior to and during DMBA ormethylnitrosourea treatment reduced mammary tumor incidence by as muchas 95% or 65%, respectively (3). Consistent with these results,supplementation of a purified diet with cabbage or broccoli, bothvegetables are good sources of I3C, also resulted in decreased mammarytumor formation in DMBA-treated rats (4). Also, in a long term feedingexperiment, in which female mice consumed synthetic diets containing I3Cat 0, 500 or 2000 p.p.m., spontaneous mammary tumor incidence andmultiplicity were significantly lower (ca. 50% reduction) at both dosesof I3C compared to untreated control animals, and tumor latency wasprolonged in the high dose group (5). I3C also has anticarcinogeniceffects on other cancer types, such as hepatic derived tissues (6-9),and can reduce benzo[a]pyrene-induced neoplasia of the forestomach (2).Because of the well documented cancer protective effects of I3C, alongwith its low toxicity, and its wide availability, this dietary indole iscurrently undergoing at least two different phase I clinical trials as acancer chemotherapeutic and preventive agent (10).

[0006] I3C has been shown to have an antiestrogenic biological activitywhen added to the diet. For example, oral administration of I3C tohumans at doses of around 500 mg daily for one week produced an increasein estradiol 2-hydroxylation of approximately 50% in both men and women(11). I3C also increased the levels of estradiol hydroxylation activityin female rats (12). I3C can, in some systems, display an antiestrogenicgrowth suppressive effect. For example, long term treatment (up to sixweeks) with 50 μM I3C blocked the estradiol-induced proliferation ofhigh density cultures (confluency for 1 weeks or longer) of human MCF7breast cancer cells (13).

[0007] A major complication in interpreting the physiological results isthat I3C is extremely unstable in acidic solution and it does notcompletely survive exposure to gastric acid (14). I3C is converted intoseveral natural indole derivatives with biological activities. The acidreaction mixture of I3C is composed of five major components which areresolvable by HPLC (15). Sensitive analytical methods reveal that l3C isconverted to several indole derivatives in acid conditions and in theintestinal contents of rats fed on a basal diet and treated orally withI3C (15-19), suggesting that these I3C derivatives may mediate theanti-estrogenic effects of I3C. I3C is converted into biologicallyactive components such as its dimer 3,3′-diindolylmethane (DIM) andindolo[3,2-b]carbazole (ICZ) through an acid-catalyzed reactionoccurring in the low-pH environment of the stomach (17). ICZ is alsoproduced, presumably from the nutritive indole, tryptophan, as ametabolic product of intestinal bacteria (19).

[0008] A general picture has emerged indicating that many, if not all,of the long term antiestrogenic biological activities of I3C result fromthe actions of one of its acid conversion products (15-19). For example,ICZ is a potent inhibitor of several estrogen-dependent responsesincluding growth inhibition of high density cultures of human breastcancer cells (20), and ICZ inhibited [³H]thymidine uptake, nuclearprogesterone and ER binding, and CAT activity in MCF7 cells transfectedwith the estrogen-responsive vit-CAT reporter plasmid. However, ICZexhibited only a very weak affinity for the estrogen receptor suggestingthat its not a direct estrogen antagonist. Further studies showed thatICZ likely mediates its antiestrogenic effects through interactions withAh (dioxin) receptor (21). ICZ competitively binds to the Ah receptor,which then translocates to the nucleus and induces P450 CYP1A1 geneexpression which has been shown to alter estrogen metabolism (21). ICZis the most potent Ah receptor agonist among the characterized I3Cderived compounds. In fact, I3C itself has a particularly low affinityfor the Ah receptor (Kd of 27 μM), compared to ICZ's high affinity (Kdof 190 pM) and the relatively moderate affinity of DIM (Kd of 90 nM) forthe Ah receptor(17). Thus, I3C per se does not mediate any of itsactivities directly through the Ah receptor, and its mechanism of signaltransduction and direct target genes are unknown. Other investigatorshave shown that the predominant I3C conversion product DIM was highlyeffective in reducing DMBA-induced mammary tumors, but DIM apparentlywas not consistently as effective as I3C (2).

[0009] We disclose herein that treatment of a human breast cancer cellswith I3C induces a reversible growth arrest in an estrogen independentmanner, resulting from a G1 cell cycle arrest. The G1 arrest in cellcycle progression correlates with a significant loss of CDK6 protein, akey cyclin dependent kinase involved in progression through the G1 phaseof the cell cycle. Our data indicate that I3C itself, and not its acidbreakdown products, is a potent anti-tumor agent, and that stablederivatives of I3C may be used to inhibit the growth ofestrogen-dependent or independent breast cancer cells and other types ofcancer cells that reveal induced CDK6.

SUMMARY OF THE INVENTION

[0010] The invention provides methods and compositions relating to novelbioactive compositions. The compositions find particular use as agentsfor inhibiting cell growth. In one embodiment, the invention providespharmaceutical compositions comprising a pharmaceutically acceptableexcipient and a compound of the general formula (I):

[0011] or a pharmaceutically acceptable salt or ester thereof, wherein:

[0012] R₁₋₅ are independently selected from the group consisting ofsubstituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroalkyl,heteroaryl, and acyl substituents wherein said compound inhibits tumorcell growth and said compound is other than indole-3-carbinol (I3C),3,3′-diinodolylmethane (DIM) and indolo[3,2-b]carbazole (ICZ).

[0013] In various preferred embodiments, at least one of R₄ and R₅ is anelectron withdrawing substituent such as ammonium, nitrile, halogen,nitro, sulfhydril, and hydroxyl groups, R₃ is an electron donatingsubstituent such as carboxylate, alkylhydroxy, alkylester andalkylether, R₂ comprises a substituted or unsubstituted aromatic moiety,and R₁ is selected from the group consisting of substituted orunsubstituted oxide, amino, carbonyl and benzyl functional groups.

[0014] In particular embodiments, the pharmaceutical compostitionscomprise a compound selected from the group consisting of a compound ofany of Tables VI-IX; a compound of the general formula (I), whereinR₁-R₅ are independently selected from H and optionally substituted(C1-C6)alkyl, (C1-C6)alkoxy, (C0-C6)acyl (C0 is formyl), halide, nitro,amino, carbonyl, hydroxyl, phenyl, benzyl, and napthyl, preferablywherein R₁ is (C1-C6)alkoxy, substituted or unsubstituted benzyl or H;R₃is (C0-C6)acyl, hydroxyethyl or H; and R₂, R₄ and R₅ are H; or apharmaceutically acceptable salt or ester thereof; and/or a compound ofthe general formula (I) wherein R₁ is alkoxy, substituted orunsubstituted benzyl or H; R₃ is acyl, (C1-C6 alkanol) or H, at leastone of R₁ and R₃ is other than H, and R₂, R₄ and R₅ are H; wherein saidcompound inhibits tumor cell growth, is other than indole-3-carbinol(I3C), 3,3′-diinodolylmethane (DIM) and indole[3,2-b]carbazole (ICZ);and a pharmaceutically acceptable excipient. In more particularembodiments of the foregoing, R₁ is alkoxy. R₁ is substituted orunsubstituted benzyl and/or R₃ is acyl.

[0015] Preferred compounds are other than a natural gastric acidmetabolite of I3C, inhibit cell growth in an estrogen-independentmanner, and particularly, by inhibiting a CDK6 activity, and demonstrateenhanced metabolic stability over I3C. The compositions mayadvantageously further comprise an antiestrogen, such as tamoxifen, ICI164384 and raloxifene.

[0016] In addition, the invention provides methods of inhibitingtargeted cell growth. In one embodiment, such methods comprisecontacting a target cell with a compound of the foregoing generalformula, or a pharmaceutically acceptable salt thereof, wherein R₁₋₅ areindependently selected from the group consisting of substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl and carbonyl; and whereinsaid compound inhibits tumor cell growth and said compound is other thanI3C, DIM and ICZ, under conditions whereby the growth the target cell isinhibited.

[0017] The invention also provides methods for evaluating the growthinhibitory activity of a compound of the foregoing general formula or anI3C derivative. In one embodiment, these methods comprise the steps ofcontacting a cell with an effective amount of the compound and measuringthe CDK6 expression in the cell, wherein a reduction in CDK6 expressioncorrelates with the growth inhibitory activity of the compound.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0018] The term substituted or unsubstituted alkyl is intended toencompass alkoxy, cycloalkyl, heteroalkyl, etc. Similarly, the termsubstituted or unsubstituted aryl is intended to encompass aryloxy,arylalkyl (including benzyl, arylalkoxy, etc.), heteroaryl, arylalkynyl,etc.; the term substituted or unsubstituted alkenyl is intended toanalogously encompass cycloalkenyl, heteroalkenyl, etc.; etc. Inprefered embodiments, the subsitituted or unsubstituted alkyl isselected from heteroalkyl (including alkoxy, etc.), cycloalkyl,acylalkyl, etc.; the subsitituted or unsubstituted alkenyl is selectedfrom heteroalkenyl, cycloalkenyl, acylalkenyl, etc.; the subsitituted orunsubstituted alkynyl is selected from heteroalkynyl, cycloalkynyl,acylalkynyl, etc.; and the subsitituted or unsubstituted aryl isselected from heteroaryl (including aryloxy, heteroaryl, heteroaryloxy,heteroarylalkyl, heteroarylalkyloxy, heteroarylheteroalkyl,heteroarylalkenyl, heteroarylalkyl and heteroaryloxyheteroalkyl, etc.),arylcycloalkyl, arylcycloalkenyl, aryloxycycloalkyl, arylalkyl,arylalkoxy, arylheteroalkyl, aryloxyalkyl, aryloxyheteroalkyl, etc.

[0019] The invention provides methods of making the subject compoundsand compositions. Generally, the compounds of the invention, includingsynthetic derivatives of I3C, are prepared by methods standard in theart of chemical synthesis, characterized for their purity, chemicalproperties and structure, then examined for their biological propertiesincluding growth suppression, effects on CDK6 protein levels andbiological stability. For example, the structures of new I3C derivativesare identified by analysis of spectral and chemical properties includinglow resolution mass spectrometry and ultraviolet spectroscopy, and themolecular formulae obtained by analysis of the high resolution massspectrum (15).

[0020] The instability ofI3C (Table I) in aqueous solutions (15, 17)derives form the fact that I3C is a vinylogous hemiaminal, and as suchit undergoes facile dehydration to the 3-methyleneindoleninium cation.This cation readily reacts with various nucleophiles including I3C.TABLE I

[0021] One preferred class of I3C derivatives with enhanced therapeuticindices is prepared by introducing substituents onto the indole nucleusthat modify the ease with which the indolenium compound is produced(Table II, class A); a second class B is modeled after metabolicproducts of I3C and related natural compounds produced in vivo; and athird class C provides modified lipid solubility of the hydroxymethylindole nucleus. Table II. TABLE II

[0022] Class A: R₁=—CH₃, —CH₂φ, —COR, OR, benzyl, etc.; R₃=—CHOHR,—CH₂R, —CH₂SO₂R, —CH₂NHR, CHO, COR, etc.

[0023] Class B: -3-methylinole oligomers; R₃=—CH₂SR, etc.

[0024] Class C: R₄ or R₂=—NO₂, —O-Methyl, —Cl, alkyl, aryl, etc.

[0025] For example, N,O-diacetyl-indole-3-carbinol (Table III) andderivatives thereofhave been found to be particularly effectiveinhibitors of cell growth. TABLE III

[0026] Several examples of the chemical synthesis of I3C derivativesfrom gramine are shown in Table IV. TABLE IV

[0027] TABLE V Exemplary compounds with multiple bioactive species(R_(x) = H, lower alkyl, phenyl, acyl). Compounds R₁ R₂ R₃ R₄ R₅ HBC001H, acyl, lower alkyl, H CH₂OR_(x) H H phenyl, methoxyl/phenoxyl HBC002H, acyl, lower alkyl, H H H acyl, nitro, phenyl, methoxyl/phenoxylhalogen HBC003 H H CH₂OR_(x) H acyl, nitro, halogen HBC004 H, acyl,lower alkyl, H H acyl, nitro, H phenyl, methoxyl/phenoxyl halogen HBC005H H CH₂OR_(x) acyl, nitro, H halogen HBC006 H, acyl, lower alkyl, H Halkyl, H phenyl, methoxyl/phenoxyl phenyl HBC007 H H CH₂OR_(x) alkyl, Hphenyl HBC008 H, acyl, lower alkyl, OR, R = lower H H H phenyl,methoxyl/phenoxyl alkyl, phenyl, acyl HBC009 H OR, R = lower CH₂OR_(x) HH alkyl, phenyl, acyl HBC010 H, acyl, lower alkyl, alkyl, phenyl H H Hphenyl, methoxyl/phenoxyl HBC011 H alkyl, phenyl CH₂OR_(x) H H HBC012 HH H acyl, nitro, acyl, nitro, halogen halogen

[0028] In a particular embodiment, R₂, R₄ and R₅ are H and R1 isselected from H, acyl, lower alkyl, phenyl, methoxyl/phenoxyl (HBC001compounds)—these are enumerated in TABLE VI HBC001 bioactive compounds.Compounds R₁ R₂ R₃ R₄ R₅ HBC001-0.1 H H CH₂OR R—H H H HBC001-0.2 H HCH₂OR R = lower alkyl H H HBC001-0.3 H H CH₂OR R = phenyl H H HBC001-0.4H H CH₂OR R = acyl H H HBC001-1.1 acyl H CH₂OR R—H H H HBC001-1.2 acyl HCH₂OR R = lower alkyl H H HBC001-1.3 acyl H CH₂OR R = phenyl H HHBC001-1.4 acyl H CH₂OR R = acyl H H HBC001-2.1 lower alkyl H CH₂OR R—HH H HBC001-2.2 lower alkyl H CH₂OR R = lower alkyl H H HBC001-2.3 loweralkyl H CH₂OR R = phenyl H H HBC001-2.4 lower alkyl H CH₂OR R = acyl H HHBC001-3.1 phenyl H CH₂OR R—H H H HBC001-3.2 phenyl H CH₂OR R = loweralkyl H H HBC001-3.3 phenyl H CH₂OR R = phenyl H H HBC001-3.4 phenyl HCH₂OR R = acyl H H HBC001-4.1 methoxyl H CH₂OR R—H H H HBC001-4.2methoxyl H CH₂OR R = lower alkyl H H HBC001-4.3 methoxyl H CH₂OR R =phenyl H H HBC001-4.4 methoxyl H CH₂OR R = acyl H H HBC001-5.1 phenoxylH CH₂OR R—H H H HBC001-5.2 phenoxyl H CH₂OR R = lower alkyl H HHBC001-5.3 phenoxyl H CH₂OR R = phenyl H H HBC001-5.4 phenoxyl H CH₂OR R= acyl H H

[0029] The methyloxyl and phenoxyl R₁ derivatives (HBC001-4 and HBC001-5compounds) exemplify a class of alkyloxides (HBC001-4 and HBC015compounds) other exemplary methoxyl/phenoxyl compounds are are shown inTable VIIA and Table VIIB. TABLE VIIA HBC001-4 and HBC020-HBC024bioactive compounds (R₃ = CH₂OH, R₄ and R₅ = H; (a)-(i) are alkyl =methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,n-pentyl and n-hexyl) R₁ R₂ H low alkyl alkanol alkyloxy benzyl methoxylHBC001- HBC021- HBC022- HBC023- HBC024- 4.1.0 4.1.0 (a)-(i) 4.1.0(a)-(i) 4.1.0 (a)-(i) 4.1.0 ethoxyl HBC001- HBC021- HBC022- HBC023-HBC024- 4.1.1 4.1.1 (a)-(i) 4.1.1 (a)-(i) 4.1.1 (a)-(i) 4.1.1 propoxylHBC001- HBC021- HBC022- HBC023- HBC024- 4.1.2.0 4.1.2.0 (a)-(i) 4.1.2.0(a)-(i) 4.1.2.0 (a)-(i) 4.1.2.0 isopropoxyl HBC001- HBC021- HBC022-HBC023- HBC024- 4.1.2.1 4.1.2.1 (a)-(i) 4.1.2.1 (a)-(i) 4.1.2.1 (a)-(i)4.1.2.1 n-butoxyl HBC001- HBC021- HBC022- HBC023- HBC024- 4.1.3.04.1.3.0 (a)-(i) 4.1.3.0 (a)-(i) 4.1.3.0 (a)-(i) 4.1.3.0 tert-butoxylHBC001- HBC021- HBC022- HBC023- HBC024- 4.1.3.1 4.1.3.1 (a)-(i) 4.1.3.1(a)-(i) 4.1.3.1 (a)-(i) 4.1.3.1 isobutoxyl HBC001- HBC021- HBC022-HBC023- HBC024- 4.1.3.2 4.1.3.2 (a)-(i) 4.1.3.2 (a)-(i) 4.1.3.2 (a)-(i)4.1.3.2 n-pentoxyl HBC001- HBC021- HBC022- HBC023- HBC024- 4.1.4 4.1.4(a)-(i) 4.1.4 (a)-(i) 4.1.4 (a)-(i) 4.1.4 n-hexyloxyl HBC001- HBC021-HBC022- HBC023- HBC024- 4.1.5 4.1.5 (a)-(i) 4.1.5 (a)-(i) 4.1.5 (a)-(i)4.1.5

[0030] TABLE VIIB HBC015-HBC020 bioactive compounds (R₂, R₄ and R₅ = H;(a)-(i) are alkyl = methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl, n-pentyl and n-hexyl) CH₂CH₂ CH₂SO₂ R₁ R₃ H lowalkyl OH OH CH₂NH₂ CH₃ methoxyl HBC01 HBC016- HBC01 HBC018- HBC01HBC020- 5-4.1.0 4.1.0 (a)-(i) 7-4.1.0 4.1.0 9-4.1.0 4.1.0 ethoxyl HBC01HBC016- HBC01 HBC018- HBC01 HBC020- 5-4.1.1 4.1.1 (a)-(i) 7-4.1.1 4.1.19-4.1.1 4.1.1 propoxyl HBC01 HBC016- HBC01 HBC018- HBC01 HBC020-5-4.1.2.0 4.1.2.0 (a)-(i) 7-4.1.2.0 4.1.2.0 9-4.1.2.0 4.1.2.0isopropoxyl HBC01 HBC016- HBC01 HBC018- HBC01 HBC020- 5-4.1.2.1 4.1.2.1(a)-(i) 7-4.1.2.1 4.1.2.1 9-4.1.2.1 4.1.2.1 n-butoxyl HBC01 HBC016-HBC01 HBC018- HBC01 HBC020- 5-4.1.3.0 4.1.3.0 (a)-(i) 7-4.1.3.0 4.1.3.09-4.1.3.0 4.1.3.0 tert-butoxyl HBC01 HBC016- HBC01 HBC018- HBC01 HBC020-5-4.1.3.1 4.1.3.1 (a)-(i) 7-4.1.3.1 4.1.3.1 9-4.1.3.1 4.1.3.1 isobutoxylHBC01 HBC016- HBC01 HBC018- HBC01 HBC020- 5-4.1.3.2 4.1.3.2 (a)-(i)7-4.1.3.2 4.1.3.2 9-4.1.3.2 4.1.3.2 n-pentoxyl HBC01 HBC016- HBC01HBC018- HBC01 HBC020- 5-4.1.4 4.1.4 (a)-(i) 7-4.1.4 4.1.4 9-4.1.4 4.1.4n-hexyloxyl HBC01 HBC016- HBC01 HBC018- HBC01 HBC020- 5-4.1.5 4.1.5(a)-(i) 7-4.1.5 4.1.5 9-4.1.5 4.1.5

[0031] Similarly, substituted and unsubstituted benzyl R₁, derivatives(HBC013 compounds) exemplify a class of aryl carbinols includingN-benzyl-indole-3 carbinol (HCB013-1.0),N-para-hydroxylbenxyl-indole-3-carbinol (HCB013-2.0) andN-para-methylbenxyl-indole-3-carbinol (HCB013-3.0)-exemplary R₃ acylcompounds of HBC013 are enumerated in Table Table VIII. TABLE VIIIHBC013 bioactive compounds. Compounds R₁ R₂ R₃ R₄ R₅ HBC013-1.0 benzyl HCH₂OR R—H H H HBC013-1.1 benzyl H CH₂OR R = lower alkyl H H HBC013-1.2benzyl H CH₂OR R = phenyl H H HBC013-1.3 benzyl H CH₂OR R = acyl H HHBC013-2.0 p-hydroxylbenzyl H CH₂OR R—H H H HBC013-2.1 p-hydroxylbenzylH CH₂OR R = lower alkyl H H HBC013-2.2 p-hydroxylbenzyl H CH₂OR R =phenyl H H HBC013-2.3 p-hydroxylbenzyl H CH₂OR R = acyl H H HBC013-3.0p-methylbenzyl H CH₂OR R—H H H HBC013-3.1 p-methylbenzyl H CH₂OR R =lower alkyl H H HBC013-3.2 p-methylbenzyl H CH₂OR R = phenyl H HHBC013-3.3 p-methylbenzyl H CH₂OR R = acyl H H HBC013-4.0o-dimethylbenzyl H CH₂OR R—H H H HBC013-4.1 o-dimethylbenzyl H CH₂OR R =lower alkyl H H HBC013-4.2 o-dimethylbenzyl H CH₂OR R = phenyl H HHBC013-4.3 o-dimethylbenzyl H CH₂OR R = acyl H H HBC013-5.0p-nitrobenzyl H CH₂OR R—H H H HBC013-5.1 p-nitrobenzyl H CH₂OR R = loweralkyl H H HBC013-5.2 p-nitrobenzyl H CH₂OR R = phenyl H H HBC013-5.3p-nitrobenzyl H CH₂OR R = acyl H H

[0032] In another embodiment, acyl (COR) R₃ derivatives (HBC014compounds), particularly wherein R═H or lower alkyl, exemplify a classof carbonyls including indole-3-carbonyl (HBC014-1.0), N-methyl-indole-3carbonyl (HBC014-3.0.0), N-ethyl-indole-3 carbonyl (HBC014-3.0.1),N-methoxy-indole-3-carbonyl (HBC013-4.0), etc.-exemplary R₃ acylcompounds of HB014 are enumerated in Table IX. TABLE IX HBC014 bioactivecompounds; (a)-(i) are alkyl = methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, tert-butyl, n-pentyl and n-hexyl Compounds R₁ R₂ R₃R₄ R₅ HBC014-1.0 H H COR R—H H H HBC014-1.1 H H COR R—CH₃ H H HBC014-1.2H H COR R = ethyl H H HBC014-1.3.0 H H COR R = n-propyl H H HBC014-1.3.1H H COR R = isopropyl H H HBC014-1.4.0 H H COR R = n-butyl H HHBC014-1.4.1 H H COR R = iso-butyl H H HBC014-1.4.2 H H COR R =tert-butyl H H HBC014-1.5 H H COR R = n-pentyl H H HBC014-1.6 H H COR R= n-hexyl H H HBC014-2.0 acyl H COR R—H H H HBC014-2.1 acyl H COR R—CH₃H H HBC014-2.2 acyl H COR R = ethyl H H HBC014-2.3.0 acyl H COR R =n-propyl H H HBC014-2.3.1 acyl H COR R = isopropyl H H HBC014-2.4.0 acylH COR R = n-butyl H H HBC014-2.4.1 acyl H COR R = iso-butyl H HHBC014-2.4.2 acyl H COR R = tert-butyl H H HBC014-2.5 acyl H COR R =n-pentyl H H HBC014-2.6 acyl H COR R = n-hexyl H H HBC014-3.0 (a)-(i)lower alkyl H COR R—H H H HBC014-3.1 lower alkyl H COR R—CH₃ H HHBC014-3.2 lower alkyl H COR R = ethyl H H HBC014-3.3.0 lower alkyl HCOR R = n-propyl H H HBC014-3.3.1 lower alkyl H COR R = isopropyl H HHBC014-3.4.0 lower alkyl H COR R = n-butyl H H HBC014-3.4.1 lower alkylH COR R = iso-butyl H H HBC014-3.4.2 lower alkyl H COR R = tert-butyl HH HBC014-3.5 lower alkyl H COR R = n-pentyl H H HBC014-3.6 lower alkyl HCOR R = n-hexyl H H HBC014-4.0 phenyl H COR R—H H H HBC014-4.1 phenyl HCOR R—CH₃ H H HBC014-4.2 phenyl H COR R = ethyl H H HBC014-4.3.0 phenylH COR R = n-propyl H H HBC014-4.3.1 phenyl H COR R = isopropyl H HHBC014-4.4.0 phenyl H COR R = n-butyl H H HBC014-4.4.1 phenyl H COR R =iso-butyl H H HBC014-4.4.2 phenyl H COR R = tert-butyl H H HBC014-4.5phenyl H COR R = n-pentyl H H HBC014-4.6 phenyl H COR R = n-hexyl H HHBC014-5.0 methoxyl H COR R—H H H HBC014-5.1 methoxyl H COR R—CH₃ H HHBC014-5.2 methoxyl H COR R = ethyl H H HBC014-5.3.0 methoxyl H COR R =n-propyl H H HBC014-5.3.1 methoxyl H COR R = isopropyl H H HBC014-5.4.0methoxyl H COR R = n-butyl H H HBC014-5.4.1 methoxyl H COR R = iso-butylH H HBC014-5.4.2 methoxyl H COR R = tert-butyl H H HBC014-5.5 methoxyl HCOR R = n-pentyl H H HBC014-5.6 methoxyl H COR R = n-hexyl H HHBC014-6.0 phenoxyl H COR R—H H H HBC014-6.1 phenoxyl H COR R—CH₃ H HHBC014-6.2 phenoxyl H COR R = ethyl H H HBC014-6.3.0 phenoxyl H COR R =n-propyl H H HBC014-6.3.1 phenoxyl H COR R = isopropyl H H HBC014-6.4.0phenoxyl H COR R = n-butyl H H HBC014-6.4.1 phenoxyl H COR R = iso-butylH H HBC014-6.4.2 phenoxyl H COR R = tert-butyl H H HBC014-6.5 phenoxyl HCOR R = n-pentyl H H HBC014-6.6 phenoxyl H COR R = n-hexyl H H

[0033] Substitution of the hydrogen atom on the indole nitrogen withelectron donating or withdrawing groups may be used to modify the rateof acid catalyzed formation of the methyleneindoleninium cation. Acylderivatives at R₁ may be prepared by treating the indole with thecorresponding anhydrides (46) and alkyl derivatives at R₁ prepared bytreating the indole-3-carboxaldehyde with alkyl halide followed by mildreduction of the product to the alcohol (47). Another method to modifyreactivity of the indole is to incorporate electron donating or electronwithdrawing groups at R₅ which affect electron density on the indolenitrogen via conjugation. Indoles substituted at this position withacyl, nitro, halogen or alkyl groups may be prepared from theappropriate phenylcarbonyl precursor used in the Fisher indole synthesisor by manipulation of appropriate available precursor indoles.Reactivity of the C-3 hydroxyl group can be modified directly by properchoice of R₃. Ether and secondary amine derivatives may be prepared bytreatment of N,O-diacetyl I3C with the desired alcohol or amine (46).C-3 secondary alcohols are available from the corresponding ketones bymild reduction.

[0034] Established in vivo products of I3C are various indole oligomers,oxidation products, and glutathione metabolites. Indole oligomers may beprepared by intermediate scale treatment of I3C with simulated gastricacid and then purification of the products by chromatography (46). Thepentacyclic aromatic derivative, ICZ, may be prepared by the Fisherindole method with 1 ,4-cyclohexadione as the carbonyl component (17).Glutathione conjugates may be prepared by treatment of I3C with thedesired sulfur derivative in basic solution (48).

[0035] An additional group of sulfur containing indole products fromBrassica plants is brassinin and related compounds (49). Brassinin maybe prepared synthetically from 3-(aminomethyl)indole by treatment withCS2 and methyliodide. Cyclobrassinin and similar compounds may beprepared from brassinin-type precursors by a pyridinium bromideperbromide mediated cyclization (50). Spirobrassinin and relatedcompounds may be prepared from brassinin or related substances bytreatment with thionyl chloride (49).

[0036] Indole derivatives with modified lipid solubilities may beprepared by appropriate choice of R₄ and R₂. Alkyl and aryl substitutionat these sites increase the lipophilicity of the product, and hydroxyalkyl substitution increase the hydrophilicity of the product, bothwithout affecting intrinsic reactivity. The products may be prepared byselection of the desired substituent in the Fisher indole precursor orvia the Vilsmeier 3-carboxaldehyde prepared from the availablesubstituted indole (47).

[0037] Occasionally, the substrates for the transformations describedherein may contain functional groups (for example, amino, hydroxy orcarboxy) which are not immediately compatible with the conditions of thegiven reaction. In such cases, these groups may be protected with asuitable protective group, and this protective group removed subsequentto the transformation to give the original functionality using wellknown procedures such as those illustrated in T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, Second Edition, John Wiley& Sons, Inc., 1991.

[0038] The compounds used as initial starting materials in thisinvention may be purchased from commercial sources or alternatively arereadily synthesized by standard procedures which are well know to thoseof ordinary skill in the art.

[0039] Some of the compounds of the invention may exist asstereoisomers, and the invention includes all active stereoisomericforms of these compounds. In the case of optically active isomers, suchcompounds may be obtained from corresponding optically active precursorsusing the procedures described above or by resolving racemic mixtures.The resolution maybe carried out using various techniques such aschromatography, repeated recrystallization of derived asymmetric salts,or derivatization, which techniques are well known to those of ordinaryskill in the art.

[0040] The compounds of the invention which are acidic or basic innature can form a wide variety of salts with various inorganic andorganic bases or acids, respectively. These salts must bepharmacologically acceptable for administration to mammals. Salts of theacidic compounds of this invention are readily prepared by treating theacid compound with an appropriate molar quantity of the chosen inorganicor organic base in an aqueous or suitable organic solvent and thenevaporating the solvent to obtain the salt. Acid addition salts of thebasic compounds of this invention can be obtained similarly by treatmentwith the desired inorganic or organic acid and subsequent solventevaporation and isolation.

[0041] The compounds of the invention may be labeled in a variety ofways. For example, labeled subject compounds find use in a variety of invitro and in vivo assays, including diagnostic assays, e.g. radioliganddisplacement assays. Accordingly, the invention provides the subjectcompounds and compositions comprising a detectable label, which may bespectroscopic (e.g. fluorescent), radioactive, etc. Similarly, thecompounds may be advantageously joined, covalently or noncovalently, toa wide variety of joined compounds which may provide pro-drugs orfunction as carriers, labels, adjuvents, coactivators, stabilizers, etc.Hence, compounds having the requisite structural limitations encompasssuch compounds joined directly or indirectly (e.g. through a linkermolecule), to such joined compounds.

[0042] The subject compositions are demonstrated to have pharmacologicalactivity in in vitro and in vivo assays, e.g. are capable ofspecifically modulating a cellular physiology to reduce an associatedpathology or provide or enhance a prophylaxis. Preferred compounds arecapable of specifically inhibiting cell growth. Established cell andanimal models to evaluate such bioactivity are well-known in the art.

[0043] Particularly suitable assays for I3C derivatives includeevaluating their ability to induce a G1 cell cycle arrest and to inhibitCDK6 protein levels in cultured MCF7 breast cancer cells. For the cellproliferative asssays, we have sucessfully employed several methods thatwe have previously described for rodent mammary tumor cells (44, 51).Briefly, human MCF7 breast cancer cells are cultured at subconfluency inmedium supplemented with 10% fetal bovine serum and 10 μ/ml insulin. Thebreast cancer cells are treated with a ranges of doses (generally 1 nMto 1 mM) of individual I3C-derivatives and I3C as a positive control andthe DIM or ICZ acid derivatives of I3C (that do not suppress growth) asnegative controls for various times up to 120 hours. The incorporationof [³H]thymidine (in a two hour pulse) into 10% trichloroacetic acidprecipitable material are used to monitor and quantitate DNA synthesisand provides a direct measure of the proliferative state of the cells.Flow cytometry analysis of nuclear DNA content after fluorescencestaining with propidium iodide is used to confirm that a given I3Cderivative induces a G1 block in cell cycle progression (44). Ourresults indicate that within the first 96 hours exposure to I3C, thebreast cancer cells show a gradual change in the DNA content of thenuclear population from one in which the cells are in all phases of thecell cycle to one in which virtually all of the mammary cells arearrested with a G1-like 2n content of DNA, whereas I3C derivatives ofenhanced efficiency require less time to induce cell cycle arrest.

[0044] Cancer cells are also treated with combinations ofindole-derivatives and either steroidal or nonsteroidal anti-estrogens,such as tamoxifen, ICI 164384 and raloxifene (39, 52), at bothsuboptimal and optimal doses to determine the ability of a given I3Cderivative to syngerize with the anti-estrogens in the growthsuppression response. Hence, the effects of the indole derivatives onestrogen receptor levels, ligand binding activity and responsiveness areexamined. For example, MCF7 cells are transiently transfected with theERE-vit-CAT reporter plasmid, which contains three estrogen responsiveelements, and the cells monitored for changes in estrogen regulated CATactivity. For comparison, the effects of the I3C derivatives on growthof the estrogen-non-responsive MDA-MB-231 breast cell line are examined.One of the characterized I3C acid products, ICZ acts as an antagonist ofAh receptor activity at low doses. Therefore, purified indolederivatives is also assayed in MCF7 cells for Ah receptor affinity,activation of Ah receptor binding to DNA, and activation of an Ahreceptor-responsive CAT reporter (17).

[0045] Our finding that I3C inhibits the protein levels of the CDK6 cellcycle component provides a unique molecular assay to determine theability of a given I3C derivative to induce a cell cycle arrest ofcancer cells. For example, 13 C derivatives may be analyzed by westernblot to determine the level of CDK6 protein produced in treated oruntreated human MCF7 breast cancer cells. In particular, cells aretreated with varying concentrations, varying times alone or in thepresence of particular sets of anti-estrogens; treatment with I3C may beused as a positive control. The cell extracts are thenelectrophoretically fractionated in SDS polyacrylamide gels, blottedonto nitrocellular membranes, the blotted proteins probed with CDK6antibodies and the CDK6 protein visualized by autoradiography. Other G1acting cell cycle genes maybe examined to confirm that a given I3Cderivative is acting similarly to I3C itself. For example, the levels ofthe other cyclin dependent kinases (CDK2 and CDK4), cell cycleactivators (cyclin D 1, D2, D3 and E) or cell cycle inhibitors (p 15,16, p21 or p27) that function at discrete times within G1 may beassessed in indole treated or untreated MCF7 breast cancer cells. Exceptfor p21 at long times of indole treatment, I3C does not significantlyalter the expression of these other cell cycle components.

[0046] Our novel observation that I3C coordinately inhibits the growthand reduces CDK6 protein levels of cultured human breast cancer cellsindicates CDK6 protein levels is a diagnostic marker for theeffectiveness of indole treatment on the suppression of the growth ofbreast cancer cells and other types of cancer cells that respond to thesynthetic I3C derivatives. For this assay, small tissue samples ofbreast tumor (or other cancer types) may be tested for CDK6 proteinlevels by indirect immunofluorescence using polyclonal or monoclonalCDK6 antibodies that recognize the human CDK6 protein. The CDK6 proteinis nuclear associated and the fluorescence signal may be observed on asingle cell level. Also, normal breast tissue can be assayed similarlysince only a few cells are needed for the assay. Alternatively, totalRNA can be extracted from small samples of tumors and RT-PCR used todetermine the presence of CDK6. However, this transcript assay requireslarger tissues samples and we have recently discoverd that the level ofCDK6 transcripts is relatively low in comparison to the protein levels.Patients with detectable CDK6 proteins are therefore candidates fortreatment with particular synthetic I3C derivatives or with combinationsof anti-estrogens and I3C derivatives if the patient's tumor cellsamples are also estrogen receptor positive.

[0047] I3C dervatives that inhibit the proliferation of MCF7 cellcultures may be tested for their ability to suppress tumor growth. Inone example of this assay, MCF7 cells are inoculated into nude athymicmice and effects on tumor growth and morphology determined as we havepreviously described for DMBA induced rodent mammary tumors (53, 54). Tomonitor the tumor formation, approximately two million cells areinjected subcutaneously into the flanks of nude athymic mice (54).Control inoculations contain saline vehicle but no cells. The inoculatedmice are either fed with particular synthetic derivatives of I3Csupplemented diets (250-2500 ppm) or chronically injected with thesynthetic indole derivatives (40-600 mg I3C/kg body weight), or with avehicle control, every 48 hours starting with the day of mammary cellinoculation. To encompass I3C derivatives which disrupt tumor formationor latency period before the tumors have reached a palpable size(approximately 0.5 cm in diameter), tumor diameters of the treated anduntreated mice are monitored twice a week using a calapiter over a sixweek time course. Depending on these results, in vivo disruption of thenormal tumor growth pattern or of the growth suppressing effects of theI3C derivatives are monitored by determining bromodeoxyuridine (BUdR)labeling index for DNA synthesis (by BUdR antibody immuno-staining). Thein vivo stability, clearance rate and tissue uptake of a particular I3Cderivative may be monitored using radioactive forms of the compounds.The growth suppressing effects of combinations of indoles and tamoxifenon MCF7-derived tumors may also be further evaluated in athymic mice.

[0048] The morphology of excised tumor cells may be analyzedhistochemically (53, 54). Residual tumors may also be dispersed assingle cell suspensions with collagenase and tested for appropriate invitro growth response to indoles on plastic substratum as well as insoft agar to monitor anchorage independent growth. In addition, thelevel of CDK6 protein may be tested in the tumor samples by westernblots or by indirect immunofluorescence.

[0049] The invention provides methods of using the subject compounds andcompositions on cells in situ (residing within the host) to treatdisease or provide medicinal prophylaxis, to downregulate CDK6expression in a cell, to reduce cell growth in vitro or in a host, etc.For use in methods applied to cells in situ, the compositions frequentlyfurther comprise a physiologically acceptable excipient and/or otherpharmaceutically active agent to form pharmaceutically acceptablecompositions. Hence, the invention provides administratively convenientformulations of the compositions including dosage units which may beincorporated into a variety of containers. The subject methods ofadministration generally involve contacting the cell with oradministering to the host an effective amount of the subject compoundsor pharmaceutically acceptable compositions. The compositions andcompounds of the invention and the pharmaceutically acceptable saltsthereof can be administered to a host in any effective way such as viaoral, parenteral or topical routes. Generally, the compounds areadministered in dosages ranging from about 2 mg up to about 2,000 mg perday, although variations will necessarily occur depending on the diseasetarget, the host, and the route of administration. Preferred dosages areadministered orally in the range of about 0.05 mg/kg to about 20 mg/kg,more preferably in the range of about 0.05 mg/kg to about 2 mg/kg, mostpreferably 0.05 to about 0.2 mg/kg of body weight per day. preferably inthe range of about 0.05 mg/kg to about 0.2 mg per kg of body weight perday.

[0050] In one embodiment, the invention provides the subject compoundscombined with a pharmaceutically acceptable excipient such as sterilesaline or other medium, gelatin, an oil, etc. to form pharmaceuticallyacceptable compositions. The compositions and/or compounds may beadministered alone or in combination with any convenient carrier,diluent, etc. and such administration may be provided in single ormultiple dosages. Useful carriers include solid, semi-solid or liquidmedia including water and non-toxic organic solvents. In anotherembodiment, the invention provides the subject compounds in the form ofa pro-drug, which can be metabolically converted to the subject compoundby the recipient host. A wide variety of pro-drug formulations are knownin the art. The compositions may be provided in any convenient formincluding tablets, capsules, lozenges, troches, hard candies, powders,sprays, creams, suppositories, etc. As such the compositions, inpharmaceutically acceptable dosage units or in bulk, may be incorporatedinto a wide variety of containers. For example, dosage units may beincluded in a variety of containers including capsules, pills, etc.

[0051] The compositions maybe advantageously combined and/or used incombination with other therapeutic or prophylactic agents, differentfrom the subject compounds. In many instances, administration inconjunction with the subject compositions enhances the efficacy of suchagents. For example, the compounds maybe advantageously used inconjuction with other anti-neoplastic agents including alkylating agentsknown in the art such as nitrogen mustards, ethylenimines andmethylmelanines, alkyl sulfonates, nitrosoureas and triazenes;antimetabolites such as folic acid analogs, pyrimidine analogs andpurine analogs; natural inhibitors such as vinca ankaloids,epipodophylotoxins, antibiotics and enzymes; homones and antagonistssuch as adrenocorico steroids, progestins, estrogens and antiestrogens,androgens and antiandrogens, gonadotropin and releasing hormone analogs;etc.; and mixtures thereof, see e.g. Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9^(th) Ed., 1996, McGraw-Hill,esp. Chabner et al., Antineoplastic Agents at pp. 1233.

[0052] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0053] I3C acts as a potent growth inhibitor of human breast cancercells in an estrogen independent manner: MCF7 human breast cancer cellswere cultured at subconfluency in medium supplemented with 10% fetalbovine serum and 10 μ/ml insulin and then treated with severalconcentrations of I3C (0, 10 μM, 30 μM, 60 μM, 100 μM and 300 μM) for 48hours. I3C strongly inhibited [³H]thymidine incorporation in MCF-7 cellcultures, which provides a direct measure of the proliferative state ofthe cells, in a dose dependent manner, with half-maximal response at 30μM I3C. At 300 μM I3C, cell proliferation was maximally inhibited andcell morphology was changed to a more elongated phenotype; however, at100 μM I3C, the cells were near maximally growth arrested but with nomorphology change. Therefore, 100 μM I3C was routinely used in theremainder of our experiments. Importantly, exposure to the twocharacterized acid products of I3C, the DIM oligomer and ICZ, failed tosuppress the growth of MCF7 cell growth. Time course studies of I3Caddition and withdrawal demonstrated that the I3C growth suppression ofMCF-7 breast cancer cells is completely reversible, demonstrating thatthis compound does not affect cell viability. Also, prolonged exposure(5 days) to I3C did not result in any detectable cell death. Analysis ofDNA synthesis over a 120 hour time course revealed that 100 μM I3Cinhibited [³H]thymidine incorporation by 80% after 72 hours and bygreater than 90% after 96 hours of treatment. Within 48 hours of I3Cwithdrawal from 48 or 72 hour I3C-treated cells, the rate of[³H]thymidine incorporation was approximately equivalent to untreatedcells.

[0054] Several lines of evidence demonstrate that I3C suppresses thegrowth of human breast cancer cells independent of any effects onestrogen receptor responsiveness. I3C dose dependently inhibited [³H]thymidine incorporation of estrogen receptor-deficient MDA-MB-231breast cancer cells. At 100 μM I3C, proliferation of these cells wasinhibited by greater than 50%. Western blot analysis using antibodies tothe human estrogen receptor confirmed that the MDA-MB-231 breast cancercells used in this assay do not express an estrogen receptor protein,while, MCF7 cells produce estrogen receptors. I3C effects on estrogenreceptor (ER) function was monitored by transient transfection of anERE-vit-CAT reporter plasmid containing the vitellogenin promoter withthree estrogen response elements linked upstream and driving thebacterial chloramphenicol acetyl transferase (CAT) gene. MCF7 breastcancer cells were transiently transfected by the lipofectamine procedureand, after 48 hours of treatment with the indicated steroids and/or I3C,reporter gene activity was assayed by monitoring the conversion of[³H]acetyl-CoA (plus unlabeled chloramphenicol) into[³H]acetylchloramphenicol using a quantitative non-chromatographicextraction procedure we have optimized (22, 23). MCF7 cells werecultured in 10% fetal bovine serum which is the conditions that weobserve the antiproliferative effects of I3C. Fetal bovine serum hasendogenous estrogen at a sufficient concentration to cause a high basallevel of reporter gene activity in MCF7 cells transiently transfectedwith the ERE-vit-CAT reporter plasmid. 100 nM estrogen stimulatedERE-vit-CAT activity, whereas, tamoxifen inhibited the basal ERE-vit-CATreporter plasmid activity by 70%. Treatment with I3C had no effect onthe estrogen receptor responsiveness of the ERE-vit-CAT activity nor didthis dietary indole modulate the antagonistic effects of tamoxifen.

[0055] I3C induces a G1 cell cycle arrest of MCF-7 breast cancer cellsand abolishes the production of the CDK6 cell cycle component: In normalmammary epithelial cells an intricate network of growth inhibitory andstimulatory signals transduced from the extracellular environmentconverge on G1 acting components which, through their concerted actions,stringently regulate cell cycle progression (24-26). The final targetsof these growth signaling pathways are specific sets of cyclin-cyclindependent kinase (CDK) complexes, which function at discrete, butoverlapping, stages of the cell cycle (24-27). Within the G1 phase ofthe cell cycle, certain cyclins (C, D1, D2, D3, E) activate the G1 CDKs(CDK2, 3, 4, and 6), while, in a complementary manner, several of thesmall proteins associated with cyclin-CDK complexes (p15, p16/Ink4,p21/Waf1/Cip1, cip2, p27) have been shown to act as specific inhibitorsof cyclin dependent kinase activity and block cell cycle progressionwithin G1 or early S phase (25, 26, 28). In contrast to normal cells,the loss of cell cycle control in G1 has been implicated in mammarytumor development and proliferation. Approximately 40% of human breastcancers show an aberrant expression and/or amplification of cyclin D1 orcyclin E (29-31). Mammary tumors can also display an inappropriateexpression and/or mutation of certain G1-acting proto-oncogenes (32,33), of growth factors and their cognate receptors which stimulateprogression through the G1 phase (34-36) and can exhibit a loss inexpression or function of certain tumor-suppressor genes (such as p53)which modulate cell cycle events late in the G1 phase (37, 38).Regulated changes in the expression and/or activity of cell cyclecomponents that act within G1 have been associated with alterations inthe proliferation rate of normal and transformed mammary epithelialcells (39-43). For example, estrogens and progesterone stimulate, andanti-estrogens inhibit, cell cycle progression of the T47D human breastcancer cell line at a point in early G1 phase of the cell cycle withcorresponding changes in cyclin D1 expression (39). Also, we haveestablished that glucocorticoids induce a G1 cell cycle arrest and alterexpression of cell cycle-regulated genes of rat tumor cells derived fromDMBA-induced mammary adenocarcinomas (44).

[0056] To assess the cell cycle effects of I3C, MCF-7 cells treated withor without 100 μM I3C for 96 hours were hypotonically lysed in thepresence of propidium iodide to fluorescently stain the nuclear DNA.Flow cytometry profiles of nuclear DNA content revealed that I3C induceda cell cycle arrest of these breast cancer cells. I3C treatment alteredthe DNA content of the MCF7 cell population from an asynchronouspopulation of growing cells in all phases of the cell cycle (29% inG1/G0; 50% in S phase and 21% in G2/M phase) to one in which most (75%)of the I3C treated breast cancer cells exhibited a 2n DNA content, whichis indicative of a G1 block in cell cycle progression. In addition,after 96 hours of I3C treatment, approximately 13% of the cells remainedwith a G2/M DNA content. Preliminary characterization of cell cyclekinetics after I3C withdrawal suggests that the primary I3C-mediatedcell cycle block occurs early in the G1 phase. This observation thatI3C, and not one of its acid breakdown products, induces a G1 cell cyclearrest of human breast cancer cells is a previously uncharacterizedgrowth response to dietary indoles.

[0057] To determine the mechanism by which I3C induces the G1 cell cyclearrest of human breast cancer cells, Western blot and Northern blotanalyses were utilized to examine whether I3C treatment regulates theprotein production and transcript expression of the cyclin dependentprotein kinases (CDKs), cyclins and CDK Inhibitor components of the cellcycle that function within the G1 phase. Most significantly, we havediscovered that I3C rapidly reduces the level of CDK6 protein within 24hours of indole treatment, which is 48 hours prior to the complete cellcycle arrest. In particular, CDK6 transcript levels dropped about 30% in5 hrs and 60% by 15 hours. In addition, dose response experimentsdemonstrated that CDK6 levels are reduced to the same extent as theinhibition of DNA synthesis implicating a causal relationship betweenthese two effects of I3C. Under these conditions, no effect on CDK2 orCDK4 expression was detected, which demonstrates the specificity of thisresponse. Furthermore, 1 3C inhibits CDK6 protein expression in estrogenreceptor negative cell lines. The existence of CDK6 is a relativelyrecent discovery (45), and our results indicate that the I3C mediatedcell cycle arrest is results from the rapid reduction in CDK6 proteinlevels. This novel observation provides a basis for a moleculardiagnostic assay to determine the sensitivity of a given tumor sample toI3C growth suppression.

[0058] Western blot analysis has shown that I3C stimulates the level ofthe p21 cell cycle inhibitor by approximately 3-4 fold only after 72-96hours of indole treatment, which is when the cells begin to displaytheir maximal cell cycle arrest. This response is not likely to initiatethe cell cycle arrest, though provides another useful molecular markerto in the examination of tissue biopsies.

[0059] Antiproliferative effects of a combination of I3C and theanti-estrogen tamoxifen on breast cancer cell growth: Estrogen receptor(ER)-containing MCF7 or ER-deficient MDA-MB-23 1 breast cancer cellswere treated for 48 hours with 100 μM I3C or 10 μM tamoxifen alone orwith a combination of both reagents, and [³H]thymidine incorporationused as a measure of cell proliferation. Tamoxifen or I3C inhibited MCF7DNA synthesis by approximately 60% and 70%, respectively, compared tovehicle controls. Interestingly, a combination both l3C and tamoxifeninhibited [³H]thymidine incorporation by greater than 90% whichrepresents a more stringent growth inhibitory effect than that observedwith exposure to either compound alone. In ER-deficient MDA-MB-23 1cells, tamoxifen had no growth inhibitory effects under conditions inwhich I3C exerted a strong growth suppression effect. The effects of acombination of tamoxifen and I3C on these ER-deficient breast cancercells were equivalent that observed with I3C added alone, which furtherimplicates that I3C can inhibit breast tumor cell growth in an estrogenindependent manner. Flow cytometry profiles of nuclear DNA contentrevealed that a combination of tamoxifen and I3C stimulated a slightlygreater percentage of MCF7 cells to arrest in G1 (85%) compared to thatobserved with either I3C (75%) or tamoxifen (70%) alone. The morestringent cell cycle arrest of MCF7 breast cancer cells by a combinationof tamoxifen and I3C is likely due to a disruption of two distinctpathways, an estrogen receptor dependent proliferative pathway disruptedby tamoxifen and an estrogen receptor-independent antiproliferativepathway induced by I3C.

[0060] Western blot analysis of the breast cancer cells treated withcombinations of I3C and/or tamoxifen demonstrated that I3C, but nottamoxifen, reduced the level of CDK6 protein. Moreover, other studieshave shown that tamoxifen reduces cyclin D1 levels in estrogenresponsive breast cancer cells (39). The selective regulation of CDK6 byI3C provides a molecular basis for the synergistic actions of acombination of I3C and tamoxifen on the growth suppression of humanbreast cancer cells since both anti-proliferative agents targetdifferent components of the cell cycle. Our observations indicate that acombination of I3C and either steroidal or nonsteroidal anti-estrogens(such as tamoxifen, ICI 164384 and raloxifene) provides and advantageouscombination hormone based therapy to control breast cancer cell growth.

[0061] References:

[0062] 1. Sharma, S., et al. (1994) Cancer Research 54: 5848-5855.

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[0116] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail byway of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A pharmaceutical compostition comprising acompound of any of Tables VI-IX and, or a pharmaceutically acceptablesalt or ester thereof, wherein said compound inhibits tumor cell growth,is other than indole-3-carbinol (I3C), 3,3′-diinodolylmethane (DIM) andindole[3,2-b]carbazole (ICZ); and a pharmaceutically acceptableexcipient.
 2. A composition according to claim 1, wherein the compoundis of Table VI.
 3. A composition according to claim 1, wherein thecompound is of Table VIIA,20 column 2 (HBC001-4 compounds).
 4. Acomposition according to claim 1, wherein the compound is of Table VIIAor Table VIIB.
 5. A composition according to claim 1, wherein thecompound is of Table VIII.
 6. A composition according to claim 1,wherein the compound is of Table IX.
 7. A composition according to claim1, wherein said compound is other than a natural gastric acid metaboliteof I3C.
 8. A composition according to claim 1, wherein said compoundinhibits cell growth in an estrogen-independent manner.
 9. A compositionaccording to claim 1, wherein said compound inhibits CDK6 activity. 10.A composition according to claim 1, wherein said compound demonstratesenhanced metabolic stability over I3C.
 11. A composition according toclaim 1 further comprising an antiestrogen.
 12. A composition accordingto claim 1 further comprising an antiestrogen selected from tamoxifen,ICI 164384 and raloxifene.
 13. A method of inhibiting cell growthcomprising contacting a target cell with a compound of claim 1 underconditions whereby the growth of said target cell is inhibited.
 14. Amethod of inhibiting cell growth comprising contacting a target cellwith a compound of claim 2 under conditions whereby the growth of saidtarget cell is inhibited.
 15. A method of inhibiting cell growthcomprising contacting a target cell with a compound of claim 3 underconditions whereby the growth of said target cell is inhibited.
 16. Amethod of inhibiting cell growth comprising contacting a target cellwith a compound of claim 4 under conditions whereby the growth of saidtarget cell is inhibited.
 17. A method of inhibiting cell growthcomprising contacting a target cell with a compound of claim 5 underconditions whereby the growth of said target cell is inhibited.
 18. Amethod of inhibiting cell growth comprising contacting a target cellwith a compound of the general formula:

or a pharmaceutically acceptable salt or ester thereof, wherein: R₁₋₅are independently selected from the group consisting of substituted orunsubstituted alkyl, alkenyl, alkynyl, and aryl; and wherein saidcompound inhibits tumor cell growth and said compound is other thanindole-3-carbinol (I3C), 3,3′-diinodolylmethane (DIM) andindole[3,2-b]carbazole (ICZ), under conditions whereby the growth saidtarget cell is inhibited.
 19. A method according to claim 18, whereinthe method is a method of inhibiting tumor cell growth comprisingcontacting a target tumor cell with, or administering to an individualin need thereof, an effective amount of an indole-3-carbinol compound ora derivative thereof, wherein said compound is stable in acidic aqueoussolution, said inhibition is estrogen-independent and said compound isnot indole-3-carbinol, 3,3′-diinodolylmethane or indole[3,2-b]carbazole(ICZ).
 20. A method for evaluating the growth inhibitory activity of anI3C deriviative, said method comprising the steps of: contacting a cellwith an effective amount of an I3C derivative; measuring the CDK6expression in said cell; and evaluating said activity, wherein thereduction in CDK6 expression is correlated with the growth inhibitoryactivity of said compound.